Immunoassay for pharmacologically active phenethylamines

ABSTRACT

New hapten compositions useful in preparing antigens which may be employed in eliciting antibodies useful in an improved radioimmunoassay for pharmacologically active phenethylamines are disclosed. Particular phenethylamins which are preferably detected by the present radioimmunoassay include the catecholamines such as norepinephrine, dopamine and epinephrine, and the amphetamines.

This is a division, of application Ser. No. 517,267 filed Oct. 23, 1974;now abandoned.

BACKGROUND OF THE INVENTION

A radioimmunoassay for catecholamines is described in U.S. Pat. No.3,704,282. The antigen utilized for eliciting the needed catecholaminespecific antibody was prepared by directly coupling the catecholamine tothe protein or polypeptide immunogenic carrier using a carbodiimidecoupling agent. The resulting antigen is formed by an amide bond linkageof the amine group of the catecholamine with pendant carboxy groups ofthe support materials. However, the antibody elicited by use of suchantigens suffers from a lack of specificity with respect to the criticalethylamine side chain.

Faraj et al., in a paper appearing Pharmacologist, June 1974, describethe preparation of an antibody specific to tyramine. The antigen usedfor eliciting this antibody is prepared by coupling p-aminohippuric acidto methylated bovine serum albumin followed by diazotization of theamino group and reaction of the diazonium intermediate with tyramine.U.S. Pat. No. 3,690,834 teaches the preparation of antigens andantibodies to a large number of biologically active compounds. Theantigens are prepared by linking the compounds to a protein carrierthrough a suitable linkage. These antigens may then be used to elicitantibodies by conventional procedures. The antibodies and spin-labeledderivatives of the biologically active compounds are then used in anassay procedure. One of the class of active compounds disclosed is theamphetamines (column 9, line 54 to column 10, line 4).

DESCRIPTION OF THE INVENTION

The present invention relates to an improved radioimmunoassay fordetection of phenethylamine compounds of the following formula: ##STR1##wherein R₁ is hydrogen or hydroxy; R₂ is hydrogen or lower alkyl; R₃ ishydrogen or lower alkyl; R₄ is hydrogen, lower alkyl, phenoxy loweralkyl and phenyl lower alkyl; R₅ is hydrogen, trifluoromethyl, hydroxyor lower alkoxy; and R₆ is hydrogen, hydroxy, halogen, or lower alkoxy.

In particular the instant invention relates to new haptenic compounds ofthe formula: ##STR2## wherein R₁, R₂, R₃, R₄ and R₅ are as above, R'₄ ishydrogen or a conventional amine protecting group and n is an integerfrom 1 to 3.

Preferred compounds for use in the practice of the present invention areobtained when R₁ is hydrogen or hydroxy, R₂ is hydrogen or methyl, R₃ ishydrogen, R₄ is hydrogen or methyl, R'₄ is t-butoxycarbonyl, R₅ ishydrogen or hydroxy and n is 1.

The t-butoxycarbonyl group is employed as a protective group incompounds of formula II to serve to prevent self-condensation reactionsduring further transformations in the preparation of the neededantigens. The t-butoxycarbonyl group can be readily cleaved to yield anantigen wherein R'₄ is hydrogen.

In order to prepare the antigens needed in the present invention, it isnecessary that the hapten of formula II be covalently bonded through thecarboxylic group to a conventional immunogenic carrier material. As usedherein, the term "immunogenic carrier material" is meant to includethose materials which have the property of independently eliciting animmunogenic response in a host animal and which can be covalentlycoupled to the above described haptens. Suitable carrier materialsinclude for example, proteins; natural or synthetic polymeric compoundssuch as polypeptides, e.g., polylysine or copolymers of amino acids;polysaccharides; and the like. Particularly preferred carrier materialsare proteins and polypeptides, especially proteins.

The identity of the protein material utilized in the preparation of anantigen of the instant invention is not critical. Examples of suitableproteins useful in the practice of this invention include mammalianserum proteins such as, for example, human gamma globulin, human serumalbumin, bovine serum albumin, methylated bovine serum albumin, rabbitserum albumin and bovine gamma globulin. Other suitable protein productswill be suggested to one skilled in the art. It is generally preferredbut not necessary that proteins be utilized which are foreign to theanimal hosts in which the resulting antigen will be employed.

The covalent coupling of the hapten to the immunogenic carrier materialcan be carried out in a manner well known in the art for establishingamide bonds. However, to ensure an adequate degree of coupling under themildest possible conditions so as to minimize any possible deleteriouseffect on the carrier material it may be desirable to convert the haptenof formula II to an isolatable activated form prior to coupling. Oneparticularly preferred isolatable activated form is theN-hydroxysuccinimide ester as indicated by formula III ##STR3## whereinR₁, R₂, R₃, R₄, R'₄, R₅ and n are as above.

Other suitable isolatable activated derivatives include thep-nitrophenyl esters; acylimidazoles; and so forth. Other methods forcoupling may be employed wherein the activated intermediates need not beisolated. Such methods include the mixed anhydride method, use of EEDQ(N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) as coupling agent andthe like.

The coupling of the hapten either as the free acid of formula II or morepreferably as an activated derivative, e.g., formula III, to theimmunogenic carrier material can be readily accomplished utilizingtechniques now well known in the art for establishing amide bonds. Thus,for example, one such technique would involve dissolving the carriermaterial and a coupling agent in a suitable inert solvent followed byadding the desired hapten of formula II. The reaction may be conductedin a temperature in the range of from about 0° C. to about 50° C.although higher or lower temperatures might be employed depending on thenature of the reactants. A most preferable temperature is about roomtemperature.

The coupling agent which may be used in the aforesaid reaction will beselected from those commonly employed in organic chemistry forinitiating amide bond formation. A particularly suitable group ofcoupling agents comprise the carbodiimides, most preferablydicyclohexylcarbodiimide or1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The molar ratio of thehapten to the carrier material will, of course, depend on the identityof the hapten utilized and the protein selected for the reaction.

Conventional conditions for the coupling reaction can be employed. Thuswhen utilizing carbodiimides as coupling agents, it is desirable toutilize a slightly acidic reaction medium for this step, e.g., a mediumhaving a pH in the range of from about 3 to 6.5, most preferably in therange of from about 4 to 6.5. Upon completion of the reaction, theexcess hapten molecules may be removed by dialysis.

As indicated previously, one preferred technique for preparing theantigens of the present invention is to first prepare and isolate anactivated derivative, i.e., a compound of formula III, and then to reactthis compound with the carrier material to form the blocked antigen.Such activated derivatives are conveniently prepared by reacting acompound of formula II with a desired activating compound, such asN-hydroxysuccinimide, and a coupling agent, such asdicyclohexylcarbodiimide, in an inert solvent. The reaction is usuallyallowed to proceed for 16-60 hours at reduced temperatures (0°-5° C.).The activated derivative may then be isolated by filtering off theby-product, dicyclohexylurea, and distilling off the solvent.

The hapten may then be coupled to the carrier material by contacting theactivated derivative with the chosen carrier material. When theactivated derivative is the N-hydroxysuccinimide ester and the carriermaterial is bovine serum albumin, this may be accomplished by adding theactivated derivative in a water-miscible solvent to an aqueous solutionof the carrier material containing a base, such as sodium bicarbonate.

Another method of coupling carrier protein to hapten (formula II) is byactivating the carboxyl group of the hapten without isolation of anintermediate and adding the activated hapten to the carrier protein. Anexample of such a reaction is the mixed anhydride obtained by reactionwith isobutylchloroformate. The hapten is dissolved in an anhydrous,water-miscible organic solvent, usually dioxane, and the solution isneutralized with an equimolar quantity of triethylamine. After stirringat room temperature, the temperature of the mixture is reduced tobetween 0° and 8° C. An equimolar quantity plus 10% excess ofisobutylchloroformate is then added and stirring is continued.Meanwhile, the carrier protein, e.g., bovine serum albumin, is dissolvedin water and the pH is adjusted to 9.0 with NaOH. The quantity ofcarrier used is equivalent to the molar quantity of hapten divided bythe theoretical number of reactive groups on the carrier. Organicsolvent is added to the carrier solution and the solution is cooled tobetween 0° and 8° C. The solution is then added to the activated haptenand coupling is allowed to proceed for 30 minutes to overnight. Thefinal ratio of organic solvent to water is 1:1.

The mixture is then adjusted to neutrality, the aqueous-organic solventis removed and aqueous solution is effected. After dialysis andlyophilization, the amine-protecting group is removed.

Following coupling of a compound of either formula II or formula III tothe carrier material, it is necessary to remove the protective group (R₄' in formulas II and III), in order to restore the free primary orsecondary amino function. In the case of the t-butoxycarbonyl protectivegroup, this may be conveniently achieved by treating the material withtrifluoroacetic acid in dichloromethane at room temperature. Therelative amounts of trifluoroacetic acid and dichloromethane and thetime duration of the treatment may be varied to suit particular cases.In general, from one to three volumes of dichloromethane per volume oftrifluoroacetic acid and reaction times of 30 to 60 minutes have beenfound to give good results.

The antigens of the present invention may be utilized to induceformation of antibodies specific to compounds of formula I above in hostanimals by injecting the antigen in such a host, preferably using anadjuvant. Improved titers can be obtained by repeated injections over aperiod of time. Suitable host animals for this purpose include mammalssuch as rabbits, horses, goats, guinea pigs, rats cows, sheep etc. Theresulting antisera will contain antibodies which will selectivelycomplex with the phenethylamines of formula I, formula II or an antigenprepared therefrom, as described above.

The specific antibodies of the present invention are useful as reagentsfor the determination of phenethylamines of formula I. In such an assay,a known amount of labelled phenethylamine is mixed with the aboveantibody and a sample containing some phenethylamine is added. Theamount of phenethylamine in the sample can be determined by measuringthe inhibition of the binding to the specific antibody of the labelledphenethylamine by the unknown sample. The reagents may be added in anyorder. A suitable assay procedure for this purpose is described ingreater detail in U.S. Pat. No. 3,709,868.

Suitable labeled phenethylamines for assay purposes includeradioisotopically labeled phenethylamines, particularly those labeledwith tritium (³ H), carbon 14 (¹⁴ C) or with iodine 125 (¹²⁵ I). One mayalso employ phenethylamines labeled with any other unique and detectablelabel such as for example an electron spin resonance group. Examples ofthe use of various electron spin resonance labeled molecules inbioassays are to be found in U.S. Pat. Nos. 3,453,288, 3,481,952 and3,507,876. Other suitable labels include chromophores, fluorophors,enzymes, red blood cells, latex particles, etc.

The novel antigens and antibodies of the present invention may beutilized in conjunction with conventional additives, buffers,stabilizers, diluents, or in combination with other physiologicallyactive substances. The preparation and use of compositions containingantigens or antibodies in conjunction with physiologically acceptableadjuvants is now well known in the art.

The preparation of haptens of formula II can readily be accomplishedstarting from a corresponding phenethylamine compound having a freehydroxy group in the para position. In one such process aspect the4-hydroxy phenethylamine derivative such as rac.4-hydroxy-alpha-methylphenethylamine, is N,O-diacylated by reaction withan acylating agent, preferably acetic anhydride. The diacyl compound isthen selectively saponified with strong base such as sodium hydroxide toyield the N-acyl compound. This product is then reacted successivelywith sodium hydride and a reagent of the formula X--(CH₂)_(n) --COORwhere n is as above, X is chloro or bromo and R is C₁₋₇ alkyl.

The N-acyl group and the ester group of the resulting phenoxyaceticester are cleaved simultaneously in refluxing caustic solution.Thereafter the free amino group is reacted with a conventional amineprotecting agent such as t-butoxycarbonyl azide. Finally, if desired thecarboxyl group is activated by the formation of an active ester such asthe N-hydroxysuccinimide ester of formula III.

In an alternative sequence the 4-hydroxyphenethylamine is convertedfirst to the N-t-butoxycarbonyl derivative as above. This derivative isthen reacted with the reagent X--(CH₂)_(n) --COOR. The ester on thelinking group is saponified as above with base and the resulting freeacid is converted to the activated ester, e.g., the N-hydroxysuccinimideester as before.

These reaction sequences as well as other aspects of the presentinvention are further illustrated in the examples. It is understood thatthe intermediates employed in preparing the antigens of the inventioninclude novel compounds, which novel compounds are included within thescope of the invention.

As used herein the term "lower alkyl" represents straight or branchedchain hydrocarbon radicals containing from 1 to 7, preferably 1 to 4carbon atoms, such as methyl, ethyl, i-propyl, t-butyl and the like.Lower alkoxy includes radicals of 1 to 7 carbon atoms such as methoxy,ethoxy, t-butoxy and the like. "Phenyl lower alkyl" includes groups suchas benzyl and phenethyl. The term "halogen" is meant to includechlorine, bromine, iodine and fluorine. Conventional amine protectinggroups include those of the acyl type (e.g., formyl, benzoyl, phthalyl,trifluoroacetyl, p-tosyl, aryl- and alkylphosphoryl, phenyl- andbenzylsulfonyl, trityl-sulfenyl, o-nitrophenylsulfenyl, -chlorobutyryland o-nitrophenoxyacetyl), of the alkyl type (e.g., trityl, benzyl andalkylidene) and of the urethane type (e.g., carbobenzoxy, p-bromo-,p-chloro- and p-methoxycarbobenzoxy, tosyloxyalkyloxy-, cyclopentyloxy-,cyclohexyloxy-, t-butyloxy, 1,1-dimethylpropyloxy,2-(p-biphenyl)-2-propyloxy- and benzylthiocarbonyl).

EXAMPLE 1 rac. N-(4-Acetoxy-alpha-methylphenethyl)acetamide

In a 1-l. flask, flushed with nitrogen and provided with a stirrer,thermometer, two dropping funnels, and a nitrogen inlet tube was placeda solution of 46.4 g. (0.2 moles) of rac.4-hydroxy-alpha-methylphenethylamine hydrobromide in 100 ml. of water.The flask was cooled in an ice bath, and when the temperature reached 5°C., 40 ml. of acetic anhydride (0.395 moles) and 440 ml. of 2N NaOH wereadded simultaneously from the two dropping funnels at approximatelyequivalent rates, maintaining the temperature below 5° C. An oilgradually separated during the addition, and the mixture was stirred foranother 4 hours at 5° C., then allowed to come to room temperatureovernight. The oil that had solidified to a mass of crystals wasrecovered by filtration. After washing with water the solid was dried invacuo over KOH and CaCl₂. Yield, 32 g. of mp. 92°-93.5°. An analyticalsample was obtained by recrystallization from ethyl acetate-- 30°-60° C.petroleum ether; mp. 97.5° - 99° C.

Microanalysis: C, 66.19; H, 7.26; N, 6.01.

Calc for C₁₃ H₁₇ NO₃ (235.26): C, 66.36; H, 7.28; N, 5.95.

The ir spectrum confirmed the presence of two acetyl groups.

EXAMPLE 2 N-(4-Hydroxy-alpha-methylphenethyl)acetamide

A 0.2 mole preparation of rac.N-(4-acetoxy-alpha-methylphenethyl)acetamide was prepared as describedabove except that a more concentrated sodium hydroxide solution wasused: i.e., 16 g. of NaOH in 50 ml. of water. Overnight, the oilpartially crystallized. The mixture was warmed to 60°-70° C. and 110 ml.of 10% NaOH solution added in portions until a permanent pH of 9 wasobtained. The mixture was cooled to 25° C., 125 ml. of n-butanol wasadded; the two phase mixture transferred to a separatory funnel, and thelayers separated. Three more extractions with n-butanol were made andthe combined extracts dried with anhydrous MgSO₄. After removal of thedrying agent by filtration, the solvent was distilled in a rotaryevaporator. The traces of n-butanol that remained were removed by theaddition of 100 ml. of water and reevaporation in a rotary evaporator,whereupon the residue crystallized on cooling; yield 46 g. The productwas recrystallized from 450 ml. of boiling water and 26 g. ofcrystalline material, m.p. 154.4°-158.5° was obtained. An analyticalsample was obtained by recrystallization from hot ethyl acetate, m.p.160.5°-163.5°.

EXAMPLE 3 Ethyl ester of rac. 4-(2-acetamidopropyl)phenoxy acetic acid

Twenty-six g. (0.135 moles) of N-(4-hydroxy-alpha-methylphenethyl)acetamide was dissolved in 400 ml. of hexamethylphosphoric triamideunder nitrogen in a 1-l., three-necked flask provided with a stirrer,thermometer, inlet tube for nitrogen and a dropping funnel. To thissolution, 4.0 g. of sodium hydride (0.167 moles) was added at T <20°,and the mixture was stirred until hydrogen evolution had substantiallyceased. To this solution of the sodium salt, 25.1 g. (0.15 moles) ofethyl bromoacetate dissolved in 25 ml. of benzene was added as rapidlyas possible; the temperature quickly climbed from 19° to 36° and thenbegan to drop. At this point the mixture remained overnight at ambienttemperature. The next morning the mixture was cautiously decomposed bythe addition of 400 ml. of ice water and the oil that separated wasrecovered by four 200-ml. extractions with ether. The combined extractswere washed several times with small portions of cold water then driedover MgSO₄. The drying agent was removed by filtration and the filtercake washed with several portions of chloroform to remove any productthat may have precipitated therewith. Distillation of the combinedchloroform and ether filtrates in a rotary evaporator left a residue of29 g. of an amber oil (77%) that crystallized on cooling. The oil wasobserved to melt at 70°-75° but was not further characterized.

EXAMPLE 4 rac. 4-(2-Aminopropyl)phenoxyacetic acid

The 29 g. of residual oil described in Example 3 above (ethyl ester ofrac. 4-(2-acetamidopropyl)phenoxyacetic acid was refluxed with 16.4 g.of sodium hydroxide (0.41 moles) in 150 ml. of water for 15 hrs., afterwhich the solution was cooled, a trace of flocculent matter removed byfiltration and the clear filtrate passed over a column of 750 ml. (0.75eq) of Dowex® 2-X4 in the hydrogen form. After washing the column withwater until the effluent pH was 4-5, the amino acid was eluted with 2 l.of 10% aqueous pyridine. Distillation of the pyridine effluent in arotary evaporator left a residue of 18 g., m.p. 301°-303° dec.

A sample was recrystallized from water after which the m.p. was303°-307° dec.

Microanalysis: C, 62.95; H, 7.28; N, 6.61.

Calc. for C₁₃ H₁₄ NO₃ (209.25): C, 63.14; H, 7.23; N, 6.69.

EXAMPLE 5 rac. 4-[2-(N-t-butoxycarbonamido)propyl]phenoxyacetic acid

To a suspension of 7.2 g. of magnesium oxide (0.18 moles) in 300 ml. ofwater was added 12.54 g. of rac. 4-(2-aminopropyl)phenoxyacetic acid andthe mixture stirred for 1 hour after which 18.0 g. of t-butoxycarbonylazide (0.126 moles) in 300 ml. of dioxane was added. The suspension wasstirred at 50° for 18 hours, after which time a pale amber suspensionresulted. The dioxane was removed by distillation in the rotaryevaporator, and the cooled aqueous residue brought to pH 3 by thedropwise addition of 10% citric acid solution. Extraction of the aqueoussolution with five 75-ml. portions of chloroform removed the acid, andthe combined extracts were washed twice with small portions of water.The extract was dried over MgSO₄, the drying agent removed, and thesolvent was distilled in the rotary evaporator. A white crystallineresidue, 16.3 g. of m.p. 142.5°-145.5° remained. A small second crop,0.46 g. of the same m.p. was obtained from the MgSO₄ filter cake.

A sample was recrystallized from toluene after which the m.p. was143°-144.5°.

Microanalysis: C, 62.12; H, 7.49; N, 4.53.

Calc. for C₁₆ H₂₃ NO₅ (309.36): C, 62.12; H, 7.44; N, 4.46.

EXAMPLE 6 t-Butyl ester of rac.N-(4-hydroxy-alpha-methylphenethyl)carbamic acid

In a 1-l., three neck, r.b. flask equipped with a stirrer, thermometer,and gas inlet tube for nitrogen were placed 46.42 g. of rac.4-hydroxy-alpha-methylphenethyl amine hydrobromide (0.2 mole), 300 ml.of water, 300 ml. of dioxane, 12 g. of magnesium oxide (0.3 mole) and 43g. of t-butoxycarbonylazide (0.3 mole) The mixture was stirred at40°-45° for 18 hours. The cooled, pale amber, turbid solution wasbrought from pH 8 to pH 5 by addition of a few ml. of acetic acid andthe dioxane distilled in a rotary evaporator. To the resulting mixtureof two liquid phases, 500 ml. of water was added and the organic phasecollected by five 100-ml. extractions with n-butanol. The combinedextracts were washed with two small portions of water, then dried overMgSO₄. After removal of the drying agent, the solvent was distilled in arotary evaporator. The last traces of n-butanol were removed by theaddition of 150 ml. of toluene and distillation in the rotaryevaporator, finally at a pot temperature of 75°. The residue, 51 g.crystallized under petroleum ether, mp. 93°-95.5°. This was dissolved in350 ml. of 60°-90° petroleum ether and 150 ml. of ethyl acetate, treatedwith a little Alox, filtered, and to the warm filtrate, 450 ml. of60°-90° petroleum ether was added. After cooling to room temperature themixture was chilled in an ice bath for 4 hours, the crystalline productcollected by filtration, washed with a little 60°-90° petroleum etherand dried. Yield 32.6 g. (65%), m.p. 98.5°-101.5°.

Microanalysis: C, 67.14; H, 8.54; N, 5.55.

Calc. for C₁₄ H₂₁ NO₃ (251.32): C, 66.91; H, 8.42; N, 5.57).

The mother liquor was stripped of solvent and covered with 100 ml. of60°-90° petroleum ether plus a few ml. of ethyl acetate. After chillingfor several hours, another 3 g. of product, m.p. 97.5°-100°, wasobtained; this was also used in the next stage.

EXAMPLE 7 Ethyl ester of rac.4-(2-t-butoxycarbonamidopropyl)phenoxyacetic acid

Thirty-two g. of the above described rac.N-(4-hydroxy-alpha-methylphenethyl) carbamic acid t-butyl ester (0.127moles) was dissolved in 350 ml. of hexamethylphosphoric triamide in a1-l., r.b. -flask provided with a stirrer, thermometer, and inlet tubefor nitrogen. To this solution, cooled to 0°-2°, was added the sodiumhydride obtained by washing 6.1 g. of the 57% dispersion in mineral oil(0.144 moles) three times with pentane. After 2 hours, hydrogenevolution became very slow, and a thick paste of the sodium saltresulted. To this suspension, 21.5 g. of ethyl bromacetate (0.128 moles)in 25 ml. of benzene was added at once; the mixture began to thin outimmediately with only a slight rise in temperature (2°). The mixture wasstirred for 2 hours at 0°-2°, then allowed to stand at room temperaturefor 12 hours. To the clear amber solution, 750 ml. of ice and water wasadded, and the oil that separated was collected by five extractions with125-ml. portions of ether. The combined extracts were freed of traces ofthe triamide by several small washes with water. After drying over MgSO₄and distillation of the solvent, there remained 43 g. of pale amber oil.One g. of this oil was distilled in a "Bantam-Ware" short path still andbut for a small forerun (solvent) the entire pot contents distilled as avery pale colored viscous oil at 163°-166°/0.03 mm.

Microanalysis: C, 64.14; H, 8.13; N, 4.40.

Calc. for C₁₈ H₂₇ NO₅ (337.43): C, 64.08; H, 8.07; N, 4.15.

EXAMPLE 8 rac. 4-(2-t-Butoxycarbonamidopropyl)phenoxyacetic acid

41 g. of the above undistilled ester, ethyl ester of rac.4-(2-t-butoxycarbonamidopropyl)phenoxyacetic acid, was heated on a steambath with 100 ml. of water and to the stirred solution 10% sodiumhydroxide was added until a permanent pH of 9 was obtained. At thispoint the hydrolysate remained at room temperature overnight. After fourextractions with 50-ml. portions of chloroform to remove neutralmaterial, the solution was cooled to 10°, and brought to pH 3 by theaddition of 25% citric acid. An oil separated that soon solidified. Theoil was dissolved by the addition of 1800 ml. of chloroform, the aqueouslayer separated, and the chloroform solution washed twice with smallportions of water and dried. After removal of the drying agent anddistillation of the solvent, there remained 35.5 g. of a white solid,m.p. 137°-139° dec. This solid was dissolved in 1 l. of hot toluene andallowed to crystallize overnight at room temperature. The product, afterdrying, first at 65° in vacuo, and then at 100°, weighed 29.1 g. m.p.144.5°-146.5°.

EXAMPLE 9 N-Hydroxysuccinimide ester of rac.4-(2-t-butoxycarbonamidopropyl)phenoxyacetic acid

To a chilled solution of 15.47 g. (0.05 moles) of rac.4-(-2-t-butoxycarbonamidopropyl)phenoxyacetic acid and 6.5 g. (0.057moles) of N-hydroxysuccinimide in 250 ml. of ethyleneglycol dimethylether was added a solution of 11.33 g. (0.055 moles) ofdicyclohexylcarbodiimide in 100 ml. of ethyleneglycol dimethyl ether.Within a few minutes the solution began to deposit dicyclohexylurea.This mixture was refrigerated for 60 hours at 4°, then thedicyclohexylurea was removed by filtration and the solvent distilledfrom the filtrate in a rotary evaporator. The residue weighed 21.1 g,m.p. 143.5°-145.5° and recrystallized from 350 ml. of 2-propanol. Afterchilling in an ice bath for 6 hours, 18 g. of crystalline solid wasrecovered, m.p. 149°-151.5°.

Microanalysis: C, 59.07; H, 6.64; N, 6.79.

Calc. for C₂₀ H₂₆ N₂ O₇ (406.44): C, 59.10; H, 6.45; N, 6.89.

EXAMPLE 10 Preparation of Immunogen (Bovine Serum Albumin Conjugate ofrac. 4-(2-aminopropyl)phenoxyacetic acid

Bovine serum albumin (BSA) (300 mg.) was dissolved in 12 ml. of waterand 6 ml. of 0.5 M sodium bicarbonate was added. TheN-hydroxysuccinimide ester (61 mg.) was dissolved in 6 ml. ofdimethoxyethane and added dropwise to the BSA solution with stirring.The solution was stirred for 4 hours at room temperature, then allowedto stand at 4° overnight. The solution was then diluted to approximately50 ml. with water and concentrated by ultrafiltration (Amicon PM-10membrane) to 5-10 ml. This dilution and concentration procedure wascarried out at least four times, or until the A₂₆₀ of the filtrate haddecreased from approximately 25 to less than 0.2. The final concentratewas dialyzed overnight at 4° against 1 liter of water. The dialysate waschanged and dialysis repeated twice for about 4 hours each time. Thesolution was then lyophilized. The lyophilized material was redissolvedin 10 ml. of trifluoroacetic acid/dichloromethane (1/l, v/v) and allowedto stand for at least 30 min. in the dark at room temperature. Thepurple solution was then evaporated to dryness under a stream ofnitrogen. The purple residue was resuspended in 20 ml. of water andbrought to pH 6-9 with 1N sodium hydroxide. The resulting clear,colorless solution was dialyzed against one liter of phosphate bufferedsaline (0.9%NaCl in 0.005 M sodium phosphate, pH 7.2) overnight at 4°.The dialysate was changed and dialysis continued for 4 hr.

The degree of incorporation of hapten in two immunogen preparations wasestimated at 30-50 moles of hapten per mole of BSA by radioimmunoassay.The molecular weight of the immunogen from one preparation was estimatedby electrophoresis on a gradient polyacrylamide gel slab in the presenceof sodium dodecyl sulfate. The average electrophoretic mobility of theimmunogen was slightly less than that of BSA and corresponded to amolecular weight of about 72,000, indicating incorporation ofapproximately 20 moles of hapten per mole of BSA, in reasonableagreement with the results of radioimmunoassay.

EXAMPLE 11 Immunization and Bleeding

For immunization of goats, the dialyzed material of Example 10 wasdiluted with phosphate buffered saline to an A₂₇₄ of approximately 1.0.The diluted immunogen was then emulsified with an equal volume ofFreund's adjuvant. The first three inoculations (using completeadjuvant) were administered at weekly intervals, the fourth afteranother 3 weeks, and monthly thereafter (the fourth and successiveinoculations used incomplete adjuvant). Each inoculation comprised twosubcutaneous injections of 0.5 ml. each.

Test bleedings were taken 2, 3, and 4 weeks after the first inoculation.After 5 weeks, and at biweekly intervals thereafter, 300 ml. of bloodwas drawn and serum prepared by standard techniques.

Preparation of Labeled rac. 4-hydroxy-alpha-methylphenethylaminehydrobromide

The radioactive amphetamine analog was prepared by iodination with ¹²⁵ Iusing techniques known per se, using Na¹²⁵ I and chloramine-T. Thesubstrate for iodination was rac. 4-hydroxy-alpha-methylphenethylaminehydrobromide (2 mg/ml. in 0.05 M sodium borate, pH 8.5).

Assay Procedure

The assay procedure is similar to that used in the Abuscreen^(TM)radioimmunoassay for morphine. The sample volume required for the assayis 0.1 ml. For quantitative evaluation, standard curves are prepared onsemilogarithmic paper using 10, 2, 1, 0.5, 0.25, 0.125 and 0.0625 μg ofd-amphetamine per ml. of normal human urine.

Performance of the Assay

Amphetamine standards were prepared by diluting a 1.363 mg/ml. aqueoussolution of d-amphetamine sulfate (1.00 mg/ml. of amphetamine as freebase) to the required concentration with normal human urine. The assaycan readily detect amphetamine levels on the order of 0.1 μg ofamphetamine in 0.1 ml. of urine.

Further, as indicated by the results summarized in Table 1, the test ishighly specific for amphetamines. Even compounds which are very closelyrelated to amphetamine in structure, such as phenylpropanolamine andphentermine show only a small fraction of the activity of amphetamine.It should be pointed out, however, that these data were obtained usingpooled antisera which were selected for low cross-reactivity. Antiseragiving a positive test (equal to or greater than the equivalent of 1μg/ml. of amphetamine) with any of the tested compounds at 10 μg/ml werenot included in this pool. The cross-reactivity of phentermine isparticularly high and common to many sera. Individual sera yielding theequivalent of 2-5 μg/ml. of amphetamine when tested with 10 μg/ml. ofphentermine are not unusual.

In Table 2, the cross-reactivity of various compounds in theradioimmunoassay is compared with those found by two other immunoassays,the Free Radical Assay Technique FRAT®) and the Enzyme MultipliedImmunoassay Technique EMIT®). The radioimmunoassay is clearly superiorin all cases which can be compared.

                                      Table 1                                     __________________________________________________________________________    CROSS-REACTIVITIES OF VARIOUS AMPHETAMINE-LIKE COMPOUNDS                      IN RADIOIMMUNOASSAY FOR AMPHETAMINE                                           __________________________________________________________________________    Compound   Structure             Cross-reactivity(1)                          __________________________________________________________________________    Phenmetrazine                                                                             ##STR4##             0.06                                         Dextromethorphan                                                                          ##STR5##             0.003                                        Phenylpropanolamine                                                                       ##STR6##             0.17                                         Propylhexedrine                                                                           ##STR7##             0.3                                          Phentermine                                                                               ##STR8##             0.7                                          Ephedrine (1-form)                                                                        ##STR9##             0.003                                        Isoproterenol                                                                             ##STR10##            0                                            Tyramine                                                                                  ##STR11##            0.17                                         Norepinephrine                                                                            ##STR12##            0.003                                        Dopamine                                                                                  ##STR13##            0.015                                        __________________________________________________________________________     (1) Cross-reactivity is defined as the concentration (in μg/ml.) of        amphetamine equivalent in the assay to 10 μg/ml. of the test drug.         Thus, for example, a test solution of 10 μg/ml. of tyramine was found      to displace from the antibody as much .sup.125 I as a solution of 0.17        μg/ml. of amphetamine.                                                

                                      Table 2                                     __________________________________________________________________________    CROSS-REACTIVITIES OF VARIOUS DRUGS IN IMMUNOASSAY TECHNIQUES                 __________________________________________________________________________             Structure                        Relative Reactivity*                Compound (if not shown in Table 1)        FRAT.sup.R **                                                                       EMIT.sup.R **                                                                      Radioimmunoassay         __________________________________________________________________________    Ephedrine                                 0.15  0.2  0.0003                   Isoxsuprine                                                                             ##STR14##                       0.05  0.2  0                        Nylidrin                                                                                ##STR15##                       0.1   --   --                       Phentermine                               1.0   --   0.07                     Propylhexedrine                           0.5   --   0.03                     Cyclopentamine                                                                          ##STR16##                       0.2   0.3  0.02                     Mephentermine                                                                           ##STR17##                       --    0.6  --                       __________________________________________________________________________     **FRAT.sup.R and EMIT.sup.R data were obtained from W. J. Braitin and I.      Sunshine, Am. J. Med. Technol. 39, 223 (1973).                                *Note that, in this table, cross-reacitvity is expressed as "relative         reactivity".                                                             

EXAMPLE 12 t-Butyl ester of N-(4-hydroxyphenethyl) carbamic acid

25 g. of tyramine (0.183 moles) and 35 g. of t-butoxy carbonylazide(0.25 moles) were stirred together in a mixture of 300 ml. of dioxane,300 ml. of water, and 4 g. of magnesium oxide (0.1 mole) at 37°-45° C.,for 21 hours under nitrogen. The dioxane was distilled from the deepamber solution in the rotary evaporator. After adjusting to pH to 6 witha little acetic acid, the solution was extracted five times with 200-ml.portions of chloroform. The combined chloroform extracts were washedwith a little water, the solution dried, then the solvent distilledleaving a syrupy residue that soon crystallized. Yield 44 g. of asomewhat tacky solid. A sample was recrystallized from ethyl acetate --60°-90° petroleum ether, after which the m.p. was 73.5°-76.5° C.

Microanalysis: C, 65.96; H, 8.39; N, 5.75.

Calc. for C₁₃ H₁₉ NO₃ : C, 65.80; H, 8.07; N, 5.90.

EXAMPLE 13 t-Butyl ester of rac. N-(4,alpha-dihydroxyphenethyl) carbamicacid

In the same manner as described in Example 12 there was obtained from47.5 g. of rac. octopamine hydrochloride (0.25 moles), 53.6 g. oft-butoxycarbonyl azide (0.375 moles) and 12 g. of magnesium oxide (0.30moles) in 1 liter of 50% aqueous dioxane, 59.0 g. of crystalline solid,of m.p. 146°-148° C. After recrystallization from a mixture of ethylacetate and 60°-90° petroleum ether, the product melted at 147°-148° C.

Microanalysis: C, 61.71; H, 7.70; N, 5.47.

Calc. for C₁₃ H₁₉ NO₄ : C, 61.65; H, 7.56; N, 5.33.

EXAMPLE 14 t-Butyl ester of rac. N-(4,alpha-dihydroxyphenethyl)-N-methyl carbamic acid

41.8 g. (0.28 moles) of rac. synephrine and 53.6 g. (0.375 moles) oft-butoxycarbonyl azide were stirred together in 1 l. of 50% aqueousdioxane in the presence of 4 g. of magnesium oxide for 22 hours at40°-45° C. The dioxane was distilled from the pale amber solution and tothe residue, 500 ml. of water was added. On chilling, 58.7 g. of solidseparated, m.p. 141.5°-143.5° (after drying at 100° for 3 hours).Recrystallization of a sample of this material gave white crystals ofm.p. 141°-141.5° C.

Microanalysis: C, 62.88; H, 7.83; N, 5.19.

Calc. for C₁₄ H₂₀ NO₄ ; C, 62.90; H, 7.92; N, 5.24.

EXAMPLE 15 Ethyl ester of 4-(2-t-butoxycarbonamidoethyl)phenoxy aceticacid

23.8 g. of the t-butoxyl ester of N-(4-hydroxyphenethyl) carbamic acid(0.1 mole) was dissolved in 250 ml. of hexamethylphosphoric triamide andto the stirred solution under nitrogen was added 2.4 g. of sodiumhydride (0.1 mole) in small portions. The mixture was stirred untilhydrogen evolution ceased (approx. 3 hours). To this stirred solutionwas added in a single portion 11.0 g. of ethyl bromoacetate (0.102moles) in 25 ml. of benzene. The external temperature rose from 7° to19° C. The mixture was approximately neutral in 10 minutes. Water andice (500 ml.) were added to the mixture, the slightly turbid mixture wasextracted five times with 150-ml. portions of ether, the combined etherextracts washed with a few small portions of water and dried overmagnesium sulfate. After removal of the drying agent, the solvent wasdistilled in the rotary evaporator. The residue, a pale amber syrup,weighed 30 g. 1 g. of this syrup was rubbed under 60°- 90° petroleumether whereupon it crystallized, m.p. 54.5°-56.5° C. Afterrecrystallization from 60°-90° petroleum ether the m.p. was 55°-57° C.

Microanalysis: C, 63.37; H, 7.79; N, 4.58.

Calc. for C₁₇ H₂₅ NO₅ : C, 63.14; H, 7.79; N, 4.33.

EXAMPLE 16 Ethyl ester of rac.4-[2-(N-t-butoxycarbonamido)-1-hydroxyethyl]phenoxyacetic acid

In the same manner as described in above in Example 15 there wasobtained from 25.3 g. of the t-butyl ester of rac.N-(4,alpha-dihydroxyphenethyl) carbamic acid, (0.1 moles) 2.4 g. ofsodium hydride (0.1 mole) in 350 ml. of hexamethylphosphoric triamide,treated with 17.0 g. of ethyl bromoacetate (0.1 mole) in 25 ml. ofbenzene, 31.7 g. of an amber syrup. A sample of this material wasdissolved in hot carbon tetrachloride and on cooling crystals weredeposited. These crystals melted at 56°-59° C., after drying in highvacuum at 45° C. the crystals were transformed to a colorless glass.

Microanalysis: C, 60.10; H, 7.32; N, 4.22.

Calc. for C₁₇ H₂₅ NO₆ : C, 60.10; H, 7.42; N, 4.13.

EXAMPLE 17 Ethyl ester of rac.4-[2-(N-t-butoxycarbonyl-N-methylamino)-1-hydroxyethyl]-phenoxyaceticacid

In the same manner as described above in Example 15 there was obtainedfrom 26.7 g. of the t-butyl ester of rac.(4,alpha-dihydroxyphenethyl)-N-methyl carbamic acid, (0.1 mole) and 2.4g. of sodium hydride (0.1 mole) in 250 ml. of hexamethylphosphorictriamide, treated with 17.0 g. of ethyl bromoacetate (0.1 mole) in 25ml. of benzene, 39 g. of viscous syrup. Since repeated efforts tocrystallize this product failed, it was hydrolyzed to the free acid asdescribed below.

EXAMPLE 18 4-(2-t-Butoxycarbonamidoethyl)phenoxyacetic acid

29 g. of the above-described corresponding ethyl ester in Example 17 wassuspended in 100 ml. of water at 80°-85° C., and the resultingsuspension treated with 10% sodium hydroxide solution (drop by drop)until a permanent pH of 9-10 was obtained. To the cooled solution a 25%solution of acetic acid was added to pH 3; whereupon a suspension ofcrystals was obtained. The solid was collected by means of sevensuccessive treatments with 100 ml. portions of chloroform. The combinedchloroform extracts were washed twice with 50 ml. portions of water,then dried. Distillation of the solvent in the rotary evaporator yielded22.3 g. of a syrup that crystallized on standing. Recrystallization ofthis solid from 125 ml. of acetonitrile yielded 15.6 g. of slightlypinkish crystals of m.p. 110°-112.5° C. Another recrystallization fromacetonitrile raised the m.p. to 112.5°-114° C.

Microanalysis: C, 61.03; H, 7.34; N, 4.63.

Calc. from C₁₅ H₂₁ NO₅ ; C, 61.00; H, 7.16; N, 4.74.

EXAMPLE 19 rac. 4-(2-t-Butoxycarbonamido-1-hydroxyethyl)phenoxyaceticacid

The above-described corresponding ethyl ester in Example 16 washydrolyzed by suspending it in water at 80°-85° C. and adding 10% sodiumhydroxide solution until a permanent pH of 9-10 was obtained. Afteracidification with 20% citric acid solution to pH 3 the solution wasextracted twice with 100 ml. portions of chloroform. The combinedchloroform extracts were dried, the drying agent removed by filtrationand the solvent distilled in the rotary evaporator. A pale amber syrupresulted. The syrup was dissolved in hot 60°-90° petroleum ether and oncooling, crystals separated. These off-color crystals, 22.55 g, weredissolved in 100 ml. of acetonitrile, treated with charcoal, filtered,and from the cooled filtrate 8.9 g. of solid, m.p. 98°-101° C. wasobtained. After one more recrystallization from acetontrile usingdecolorizing carbon 6.65 g. of slightly grayish crystals was obtained,m.p. 109°-111° C.

Microanalysis: C, 57.90; H, 6.88; N, 4.85.

Calc. for C₁₅ H₂₁ NO₆ : C, 57.87; H, 6.80; N, 4.50.

EXAMPLE 20 rac. 4-[2-(N-t-Butoxycarbonyl-N-methylamino)-1-hydroxyethyl]phenoxyacetic acid

The corresponding ethyl ester of Example 17 was subjected to hydrolysisas described above in Examples 18 and 19. However, despite all attemptsit was not possible to obtain the acid in crystalline form. It wastherefore converted to the crystalline S-benzylthiuronium salt in thefollowing manner: A weighed sample of the syrupy acid was treated withsufficient sodium hydroxide to form the sodium salt (pH 7.5-8). To thissolution was added a concentrated solution of S-benzylthiuroniumchloride, whereupon a precipitate formed. The solid was recovered,washed with cold water and then recrystallized from water to obtainwhite crystals of m.p. 163°-165° C. Analysis indicated that the crystalswere the S-benzylthiuronium salt of the above-named acid.

Microanalysis: C, 58.64;, H, 6.77; N, 8.55.

Calc. for C₁₆ H₂₂ NO₆.C₈ H₁₁ SN₂ : C, 58.60; H, 6.77; N, 8.60.

EXAMPLE 21 N-Hydroxysuccinimide ester of rac.4-[2-N-t-butoxycarbonyl-N-methylamino)-1-hydroxyethyl]phenoxyacetic acid

3.25 g. (0.01 moles) of the syrupy acid described above in Example 20was dissolved in 40 ml. of dimethoxyethane together with 1.15 g. ofN-hydroxysuccinimide (0.01 moles) and to this solution was added 2.06 g.of dicyclohexylcarbodiimide (0.01 mole) whereupon the solution warmedpreceptibly. The mixture was then stored at 5° C. for 21 hours. Thedicyclohexylurea that had separated was removed by filtration, thefilter cake washed with a little dimethoxyethane, and the combinedfiltrates distilled in the rotary evaporator. There remained 3.70 g. ofa turbid syrup that was dissolved in 75 ml. of toluene and theundissolved dicyclohexylurea that had separated was removed byfiltration. Distillation of the solvent left a syrup that was dissolvedin 50 ml. of 2-propanol; 60°-90° petroleum ether was added to turbidityand the solution stored at 5° C. for 5 days. The crystals that separatedwere recovered, yield 2.5 g. of m.p. 106°-5° C.

Microanalysis: C, 56.58; H, 6.38; N, 6.73.

Calc. for C₂₀ H₂₆ N₂ O₂ : C, 56.87; H, 6.20; N, 6.63.

EXAMPLE 22

The N-hydroxysuccinimide ester of rac.4-[2-(N-t-butoxycarbonyl-N-methylamino)-1-hydroxyethyl]phenoxy aceticacid was coupled to bovine serum album (BSA) following the procedure setforth below.

A total of 300 mg. (0.00447 mmoles) of bovine serum albumin (BSA) in 12ml. of water was treated with 6 ml. of a 0.5 M solution of sodiumbicarbonate and then with 6 ml. of dimethoxyethane containing 60 mg.(0.075 mmoles) of the N-hydroxy-succinimide ester of rac.4-[2-(N-t-butoxycarbonyl-N-methylamino)-1-hydroxyethyl]phenoxy aceticacid. The mixture was stirred for 3 hours at room temperature, 96 ml. ofethanol was added and the solution evaporated to a small volume. Theresidue was then dialyzed against 200 volumes of water for 3 days withtwo changes per day and then the protected antigen was lyophilized. Asecond run was carried out under the same conditions as above with theexception that 162 mg. of the activated ester was employed.

Removal of the t-butoxycarbonyl protecting group was accomplished bystirring 50 mg. of the protected antigen with 50% trifluoroacetic acidin 50 ml. of methylene chloride for 1 hour at room temperature. Thetrifluoroacetic acid was then removed by flash evaporation. The residuewas washed with water followed by evaporation. The residue was thentaken up in water and lyophilized to yield the desired antigen.

Examination of the antigen produced from both runs using proteinanalysis and differential U.V. spectral analysis indicated that theantigen of run 1 contained 14 moles of hapten per mole of BSA (17%substitution based on 85 theoretically available amino groups) while theantigen of run 2 contained 25 moles of hapten per mole of BSA (29%substitution). This antigen is used to elicit antibody specific forepinephrine, metanephrine and synephrine. The antibody may be used inthe assay of Example 14 using the radiolabeled compounds epinephrine H³,synephrine ¹²⁵ I. and metanephrine ¹²⁵ I. The tritiated compound iscommercially available and the iodinated labels are prepared using thestandard chloramine T method well known in the art.

EXAMPLE 23

Rac. 4-(2-t-butoxycarbonamido-1-hydroxyethyl)phenoxy acetic acid wascoupled to BSA utilizing the mixed anhydride method as follows.

A total of 39.46 mg. (0.1269 mmoles) of the protected hapten was addedto 1 ml. of dry dioxane followed by the addition of 0.1269 mmoles oftriethylamine in 0.5 ml. dioxane. The mixture was stirred at roomtemperature for 10 minutes and then cooled to 8° C. 0.1395 mmoles ofisobutylchloroformate in 0.5 ml. dioxane was added and the solution wasstirred for 20 minutes.

In a separate flask 100 mg. of BSA is dissolved in 10 ml. of water, thepH was adjusted to 9 with sodium hydroxide and 8 ml. of dioxane wasadded slowly with stirring. The solution was cooled to 8° C. and theprotected hapten solution from above was added and stirred 30 minutes at8° C. and then overnight at 4° C. at pH 9.

The solution was then treated with acid to neutrality, the solventremoved and the residue taken up in 5 ml. water (NaOH added to effectsolution). Five milliliters of this solution was dialyzed successivelyagainst 6,000 ml. each of 0.5N NaOH, 0.1N NaOH, and H₂ O (2times). Theamine-blocked antigen solution was removed and lyophilized. Thet-butoxycarbonyl group was removed with trifluoroacetic acid asdescribed in Example 22.

Analysis of the resulting antigen by protein determination and U.V.analysis indicated that there were 64 moles of hapten per mole of BSA.The antigen so produced is useful in eliciting antibodies whichrecognize norepinephrine, normetanephrine and octopamine when injectedinto suitable animals. Such antibody may be incorporated in the assay ofExample 14 using radiolabeled norepinephrine ³ H, normetanephrine ³ H or¹²⁵ I and octopamine ¹²⁵ I. The labeled compounds are prepared as inExample 22.

EXAMPLE 24

The procedure of Example 23 was employed utilizing4-(2-t-butoxycarbonamidoethyl)phenoxyacetic acid as hapten to produce anantigen containing 69 molecules of hapten per molecule of BSA. Thisantigen is useful in eliciting antibodies for dopamine and tyramine. Theantibody may be used in the assay of Example 14 with dopamine ³ H andtyramine ¹²⁵ I, the source of which is as in Example 22.

EXAMPLE 25N-[2-(4-hydroxyphenyl)ethyl]-2-[4-(2-aminopropyl)-phenoxy]acetamide

a. Into a 100 ml. flask equipped for stirring, with thermometer, andunder nitrogen were placed 1.8 g. (5.8 × 10.sup.⁻³ mole) ofDL-3-[2-(t-butoxycarbamido)-propyl]phenoxyacetic acid, 45 ml. of drytetrahydrofuran and 1.0 g (6.05 × 10.sup.⁻³ mole) of carbonyldiimidazole. The mixture was stirred at room temperature (21° C.) for 2hours when 0.63 g. (4.6 × 10.sup.⁻³ mole) of tyramine was added.Stirring was continued at 21° C. overnight (ca. 18 hours). The mixturewas then transferred to a separatory funnel and diluted with severalvolumes of ethyl acetate. The mixture was shaken with 3 portions ofwater. The organic phase was separated, dried over anhydrous sodiumsulfate and evaporated at reduced pressure to give 2.6 g. ofN-[2-(4-hydroxyphenyl)ethyl]-2-[4-t-butoxycarbonamidopropyl)-phenoxy]acetamide,identified by nuclear magnetic resonance spectroscopy.

b. Into a 100 ml. flask equipped with thermometer, stirrer, and undernitrogen, were placed 1.97 g. of the product of step (a) and 28.4 ml. oftrifluoroacetic acid. After stirring for 1 hour at room temperature (21°C.), the mixture was concentrated at 40° C. The residue was dissolved in15 ml. of water and placed on a 15 mm × 150 mm column of ion exchangeresin (IR-4B, acetate form) and eluted with water. Approximately 10 15ml. fractions were collected, containing only the desired material.Evaporation of the combined fractions left a residue of 2.5 g. which wasdissolved in water, neutralized by addition of solid sodium bicarbonateand stored in the refrigerator until precipitation was complete. Afterwashing with cold water and crystallization, 0.4 g., m.p. 160°-163° C.was obtained. By further cooling, and concentrations, additional productwas obtained to total 0.52 g. ofN-[2-(4-hydroxyphenyl)ethyl]-2-[4-(2-aminopropyl)-phenoxy]acetamide.

Anal. Calcd. for C₁₉ H₂₄ N₂ O₃ ; C, 69.49; H, 7.37; N, 8.53. Found: C,69.41; H, 7.46; N, 8.40.

NMR spectrum: Compatible.

EXAMPLE 26

The N-[2-(4-hydroxyphenyl)ethyl]-2-[4-(2-aminopropyl)-phenoxy]acetamidewas iodinated by procedures known per se employing Na ¹²⁵ I andchloramine-T to giveN-[2-(4-hydroxyphenyl)ethyl]-2-[4-(2-aminopropyl)-phenoxy]acetamide-¹25I.

We claim:
 1. N-[2-(4-hydroxyphenyl)ethyl]-2-[4-(2-aminopropyl)-phenoxy]acetamide. 2.N-[2-(4-hydroxyphenyl)ethyl]-2-[4-(2-aminopropyl)-phenoxy]acetamide-.sup.125I.